Project Summary (Abstract) Preventing rebound of HIV following antiretroviral therapy interruption (ATI) remains a major problem. However, pinpointing the exact cells that serve as reservoirs from which the virus rebounds has proven elusive. An- tiretroviral therapy (ART) has shown effectiveness in removing productively infected cells from the periphery, with limited success in brain. However, once ART is withdrawn, virus rebounds, and infection in the brain coin- cide with neuroinvasion and concomitant neuroinflammation. The Long-Term Goal is to establish a basis for identifying and then preventing HIV rebounding upon ATI. The Objective of this Application is to develop a strategy for identifying, and a means of measuring, the myeloid reservoir in the CNS and other relevant organs. The Central Hypothesis is that infected microglia and other myeloid cells would be the source of rebounding virus following ATI. This is based on previous work from the interdisciplinary team of experience with ART and reservoir research in macaques (Ling), neuroimmunology (MacLean) and HIV-1/SIV molecular virology (Ling, Maness & Keele). We will employ a barcoded SIV (Keele), advanced imaging techniques, cell culture and next generation sequencing of the barcoded region to identify latently infected cells and determine which cells are the source of rebound virus upon ATI. Two Specific Aims are proposed: Specific Aim 1: Determine whether myeloid cells in CNS harbor reactivatable replication-competent virus in SIV-infected macaques under fully suppressive ART. While lymphocytes receive the most attention for HIV infection, myeloid cells in brain, lungs and lymphoid tissues are often overlooked as founder/ refounder target cells of HIV infection. Our hy- pothesis here is that SIV infected myeloid cells can be reactivated to produce infectious virus for new infection. Specific Aim 2: Determine whether myeloid cells in the CNS are a source of rebound virus upon ART interruption (ATI). We hypothesize that microglia and other myeloid cells would be the source of virus re- bound upon ATI. Our central hypothesis for this aim is that brain-specific barcodes and evolved sequences will be identified within myeloid cells following ATI and have high similarity with sequences isolated from rebound virus in peripheral blood. We believe once this project is completed, it will be possible to identify target cells to prevent HIV rebounding in the absence of continued ART. !